Project 5 Abstract Adoptive immunotherapy with antigen-specific T cells or T cells engineered to express a chimeric antigen receptor (CAR) has emerged as a potentially curative approach to the treatment of drug refractory viral infections and relapses of B lineage malignancies following allogeneic hematopoietic cell transplants (HCT). Our program has recently provided evidence that EBV-specific, appropriately HLA restricted T cells from HLA partially matched third party donors can also induce durable complete or partial remissions in 70% of allo-HCT recipients and 61% of solid organ transplant (SOT) recipients with Rituxan resistant and, in SOT patients, chemotherapy refractory EBV+ lymphomas. Consistent with our demonstration that alloreactive T-cells are depleted in the course of repeated in vitro sensitizations with autologous EBV+ BLCL, adoptive transfer of 3rd party EBVCTL has not been associated with either impairment of allograft function or GVHD. Strikingly, cytokine release syndrome has also not been observed. In this project, we propose strategies to extend and enhance the application of banked third party donor- derived EBV-specific CTLs that currently can provide effective and immediately accessible, ?off the shelf? adoptive therapies for alloHCT and SOT patients, to patients with other EBV-associated malignancies including hematologic diseases such as EBV+ Hodgkins disease, NK T cell lymphomas, HLH and Burkitt lymphomas as well as EBV negative B cell lineage leukemias and lymphomas for which an alloHCT is indicated. The project will test in vitro and in preclinical models, three hypotheses: 1) Epigenetic modifiers such as 5-azacytidine, decitabine and the HDAC inhibitors, vorinostat and romidepsin which have been found to induce latently infected EBV+ malignancies to express lytic cycle genes of EBV so as to render them susceptible to ganciclovir can be used in repeated short exposures of low toxicity to induce latency I and II malignancies, such as Burkitt lymphomas, NK T cell lymphomas, EBV+ Hodgkins disease and nasopharyngeal carcinoma, to express latency 3 and early lytic EBV proteins such as BZLF-1, thereby rendering them susceptible to EBV-specific, HLA- restricted 3rd party CTLs sensitized with autologous EBV transformed BLCLs that predominantly contain T cells specific for these EBV antigens; 2) A limited bank of 3rd party CD19 CAR+ EBVCTL can provide immediate and effective adoptive therapy for most patients relapsing with CD19+ B lineage ALL or DLBCL post transplant without GVHD. Furthermore, their anti-tumor activity and persistence can be further augmented by CARS directing the expression of IL-12. 3) The anti-tumor activity of the CD19 CAR+ EBVCTLs and CD19 CAR+ EBVCTLs secreting IL-12 can be further increased by a) pre-infusion treatment with low doses of epigenetic modifiers so as to alter tumor expression of activating and inhibitory ligands, and thereby enhance their sensitivity to the CD19 CAR+, EBVCTLs, and/or b) co-administration of a checkpoint inhibitory to enhance the capacity of the CD19 CAR+ EBVCTLs to engage and lyse B lineage ALL and DLBCL.